The primary focus of the research in this proposal will be directed towards elucidation of the physiological roles of phosphorylation of specific myofibrillar proteins. Troponin is readily phosphorylated by cyclic AMP-dependent protein kinase while myosin is phosphorylated by a calcium-dependent myosin light chain kinase. These investigations will examine the relationships between the phosphorylated states of these proteins and myocardial performance and, in addition, involve research on purified proteins in order to relate the biochemical properties of these phosphorylation reactions in vitro to their regulatory properties in vivo. The temporal and dose response relationships between extent of troponin-I phosphorylation and left ventricular maximum positive and negative dP/dt will be examined in isolated, working hearts after isoproterenol, and other pharmacological agents which affect cyclic AMP formation. Phosphorylation of myosin light chain will be examined in single papillary muscles and perfused hearts under conditions that alter cytoplasmic Ca2 ion fluxes. These measurements will be made under steady state conditions as well as during discrete portions of the contraction cycle. The possible alteration of these reactions by chronic perturbations (exercise-induced myocardial hypertrophy, glucocorticoids and thyroid hormone) will also be examined. Myosin light chain kinase will be purified from ventricular muscle and its enzymatic and physical properties will be compared to the other isozymic forms from skeletal and smooth muscles. Limited proteolysis of myosin and troponin-I will be used to assess changes in protein conformations and interactions with other myofibrillar proteins due to phosphorylation. Thus, these studies will provide important information on the roles of myofibrillar protein phosphorylation in relation to the physiological performance of the heart and altered responsiveness to pharmacological and pathophysiological perturbations.